Purification,Characterization Of Anti-Alicyclobacillus Bacteriocins Produced By Lactic Acid Bacteria

A.acidoterrestris is considered to be one of the most important target organisms in quality control of fruit juices and fruit juice-containing drinks,because of its ability to germinate and outgrow spores under high acid conditions.Therefore,developing an effective method for inhibiting or controlling the germination and outgrowth of A.acidoterrestris in such products is an urgent need of the beverage industries.The use of bacteriocins from lactic acid bacteria for food preservation has been a matter of extensive work in recent years,as most of them are considered to be probiotics since they exert health effects beyond basic nutrition when ingested in sufficient numbers.Application of bacteriocins in preservation of fruit juices has seldom been studied.Bacteriocins produced by lactic acid bacteria are generally stable at high temperature and low pH value;and hence it might be suitable for inhibiting the growth of food-borne pathogens,and spoilage bacteria in acidic fruit juices drinks.Purpose of this work was to primarily investigated the anti-Alicyclobacillus bacteriocin.The main results are as following:(1)Isolated three strains out of 20 strains of lactic acid bacteria.All three strains were shown the abilities to produced anti-Alicyclobacillus peptides.Several protein purification methods were tested and optimum purification procedures were designed for each bacteriocin.(2)Bacteriocin produced by B.animalis subsp.animalis C6165 was purified by ammonium sulfate precipitation associated with SP-Sephorase and reverse-HPLC.Bacteriocins produced by L.paracasei CICC 20241,L.rhamnosu CICC 20975 were purified by adsorption and disadsorption from cells of the producer strains associated with SP-sephorase and reverse-HPLC.N-amino acid sequencing results were subjected to homology analysis.And bacteriocins from B.animalis subsp.animalis and L.paracasei CICC 20241 were considered as newly discovered bacteriocins,known as bificin C6165 and paracin C,respectively.Partial amino acid sequencing of bacteriocin from L.rhamnosu CICC 20975,named as bacteriocin RC20975,was found to be significantly similar with Acidocin A,a class ? bacteriocin.These bacteriocins were active in acid environment and heat stable,except for bacteriocin RC20975.(3)The primary mode of action of bacteriocins were most probably due to pore formation,as indicated by the efflux of K+ from metabolically active cells of Alicyclobacillus acidoterrestris.Interestingly,bacteriocin discovered from B.animalis subsp.animalis C6165 was seldom to be reported.So I focused on bificin C6165 and investigated the application,gene and protein structure and fennentation of this bacteriocin in further study.(4)Analysis of the structural gene bif A revealed that bificin c6165 was synthesized as a 56-amino-acid precursor including a 23-amino-acid N-terminal extension.The molecular mass was determined as 3395 Da.The difference of molecular mass between the test result from the MALDI-TOF mass and the predicted value from bif meaning that Three H2O molecular was lost during the modification procedure on bificin C6165.Other physic/chemic characteristic of bificin C6165 were studied with bioinformatics tech.(5)Although the antimicrobial ability reduced,bificin C6165 was active in commercial fruit juices.Unfortunately,this bacteriocin wasn't active against spores of Alicyclobacillus acidoterrestris in commercial apple juice,even though it works well in three other commercial fruit juice.MTT test result indicated that bificin C6165 don't have cytotoxicity.However,bificin C6165 was able to increase heat sensitivity of spores in apple juice.The heat resistant of spores showed as D value reduced as addition of bificin C6165.Apple juice quality test indicated that the fruit juice was satisfied for Chinese National Production Standard,even if the color and transmittance was decreased.Additionally,encapsulation of bificin C6165 was a promising way for application of this bacteriocin.(6)Finally,sorts of medium and medium components were investigated on the production of bificin C6165.pH,temperature and inoculation size dependent property were observed when B.animalis subsp.animalis C6165 produced Bificin C6165.QS system for regulation Bificin C6165 biosynthesis was identified.Latter then,the kinetics model of fermentation of B.animalis subsp.animalis C6165 were investigated.The results showed that bacteria growth presents typical S curve model with is described with Logistic equation;Bificin C6165 production and the bacteria growth are coupling,so it is described with logistic equation as well;addtionaly,substrate consumption kinetics Processes were described with the Luedeking-Piret equation,...